Sedimentary pigments are useful proxy indicators of phytoplankton biomass, community structure, primary productivity and human influence in lacustrine and oceanic ecosystems. Pigments are relatively labile due to their complex chemical structures, which makes the extraction and analysis of sedimentary pigments challenging. In addition, it is important to select appropriate methods to study sedimentary pigments in regions which lack previous investigations. In this study, we adopted the L-9(3(4)) orthogonal design to develop methods of extraction and HPLC (high performance liquid chromatography) analysis of sedimentary pigments at two lakes on the Tibetan Plateau: meromictic lake-Dagze Co and dimictic lake-Jiang Co. The orthogonal design comprises 9 combinations of various parameters for extraction and HPLC analysis. The results show that the type and volume of solvent are the most important factors for pigment extraction, and the mobile phase and column selection are the most important for HPLC analysis. For the study sites, we found that the best methods to extract sedimentary pigments are as follows: the use of a mixture of acetone:methanol:water (80:15:5, v:v:v) as the extraction solvent; solvent/ sample ratio of 10 mL/g; sonication for 30 s and standing extraction for 6 h. The best methods for HPLC analysis are as follows: Zorbax Eclipse plus C8 column with mobile phase A, methanol:acetonitrile:0.25 M aqueous pyridine (50:25:25, v:v:v) and mobile phase B, methanol: acetonitrile: acetone (20:60:20, v:v:v); pH of mobile phase A adjusted to 6 with acetic acid; and HPLC column temperature maintained at 40A degrees C. The study provides an experimental basis for future investigations of past changes in primary productivity and the response of lake ecosystems to climate change and human activities on the Tibetan Plateau.